We study regulatory mechanisms involved in nutrient control of gene expression in the budding yeast Saccharomyces, focusing on a system (general amino acid control) that induces genes encoding amino acid biosynthetic enzymes in response to starvation for any amino acid. The transcriptional activator in this pathway, GCN4, is induced at the translational level in starved cells by phosphorylation of translation initiation factor 2 (eIF2) by the protein kinase (PK) GCN2. Phosphorylation of eIF2 reduces the concentration of the ternary complex (TC), containing eIF2, GTP, and initiator methionyl-tRNA, which transfers tRNAiMet to the 40S ribosome in producing the 43S preinititiation complex (PIC). The inhibition of TC assembly impedes general protein synthesis but induces GCN4 translation by a reinitiation mechanism involving small upstream open reading frames (uORFs) in the GCN4 mRNA leader. A reduction in TC levels allows 40S ribosomes scanning the GCN4 mRNA leader after translating uORF1 to bypass uORFs 2-4 and reinitiate at the GCN4 start codon instead.[unreadable] Previous biochemical analysis of translation initiation suggested that eIF1, eIF1A and eIF3 promote TC binding to the 40S subunit, but there was no evidence for these requirements in vivo. We hypothesized that, if eIF1A is required for efficient TC recruitment, it should be possible to identify eIF1A mutations that mimic the effect of eIF2 phosphorylation and derepress GCN4 translation in gcn2 cells (Gcd- phenotype). eIF1A contains an OB-fold present in bacterial IF1 plus N- and C-terminal extensions not present in IF1. We found that truncating the C-terminus (?'C) or mutating OB-fold residues (66-70) of eIF1A reduced general translation in vivo but increased GCN4 translation (Gcd- phenotype) in a manner suppressed by overexpressing TC. Consistent with this, both mutations diminished steady-state binding of TC, eIF5 and eIF3 to native PICs in cell extracts, and ?'C reduced the rate of TC binding to 40S subunits in vitro in reactions with purified components. The assembly defects of the OB-fold mutation can be attributed to reduced 40S-binding of eIF1A, whereas ?'C impairs eIF1A function on the ribosome. A substitution in the C-terminal helix (98-101) also reduced 43S PIC assembly in vivo. Rather than producing a Gcd- phenotype, however, 98-101 impairs GCN4 derepression in a manner consistent with defective scanning by reinitiating ribosomes. Indeed, 98-101 allows formation of aberrant 48S complexes at non-AUG codons in an in vitro assay for scanning, and it increases initiation at UUG codons in vivo. Thus, the OB-fold is crucial for ribosome-binding and the C-terminal domain of eIF1A has eukaryotic-specific functions in TC recruitment and scanning.[unreadable] Previously, we showed that the eIF3 complex, eIF1 and eIF5 reside with TC in a multifactor complex (MFC) and we mapped interactions between these factors that stabilize the MFC. The N-terminal domain (NTD) of eIF3c/NIP1 interacts directly with eIF1 and eIF5 and indirectly with the TC via eIF5. We showed previously that mutating two 10-amino acid clusters in the NIP1 NTD confers a Gcd- phenotype that is suppressed by overexpressing TC and also decreases PIC assembly in vivo. These findings provided evidence that interactions of eIF3c/NIP1 with eIF1, eIF5 and TC enhance PIC assembly in vivo. To provide a more exhaustive test of the importance of MFC formation, and to evaluate the relative importance of eIF3, eIF2 and eIF5 in 43S complex assembly in vivo, we determined the effects of depleting each of these factors on association of all other MFC constituents with native PICs. Using !?degron!?mutants endowed with conditional expression of eIF2?O, eIF3a plus eIF3b, or eIF5, we found that depletion of each factor reduced 40S binding of all MFC constituents, indicating that eIF2, eIF3 and eIF5 are interdependent for optimal 40S binding in vivo. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. However, whereas depleting eIF3 impaired mRNA binding to 40S subunits, depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, eIF3 can function independently of eIF4G to promote binding of at least some mRNAs to native PICs, and it appears that eIF4G has a rate-limiting function downstream of mRNA recruitment in vivo.[unreadable] Most ATP-binding cassette (ABC) proteins function in membrane transport, but yeast encodes a family of soluble ABC proteins that includes translation elongation factor 3 and GCN20. We showed previously that GCN20 acts on the ribosome to stimulate activation of protein kinase GCN2 by uncharged tRNA. Subsequently, we found that the soluble ABC protein RLI1 interacts with the MFC, stimulates 43S PIC assembly, and enhances translation initiation in yeast cells. We recently assisted Michael Dean!|s group (NCI) in showing that the human RLI1 has similar functions in translation initiation. Independently, we discovered that the soluble ABC protein ARB1 shuttles from nucleus to cytoplasm and participates in ribosome biogenesis. We showed that depleting ARB1 leads to a deficit in 40S subunits by delaying the processing of 35S and 20S pre-rRNAs needed to produce mature 18S rRNA. Depleting ARB1 also delays rRNA processing in the 60S biogenesis pathway. We found that ARB1 co-sediments with 40S, 60S and 80S/90S ribosomal species and is associated with other proteins (TIF6, LSG1) implicated in 60S or 40S biogenesis. We propose that ARB1 functions as a mechanochemical ATPase in the 40S and 60S ribosomal biogenesis pathways. Together with our previous analysis of GCN20 and RLI1, and preliminary analysis of soluble ABC protein NEW1, it now appears that all ABC proteins in the eEF3/GCN20 family have functions connected with ribosomes and protein synthesis.[unreadable] We previously established that optimal transcriptional activation of biosynthetic genes by GCN4 requires multiple coactivators (SAGA, SWI/SNF, Mediator, RSC) that are recruited by GCN4 to its target genes and stimulate PIC assembly at the promoter. The Paf1 complex (Paf1C) interacts with RNA Polymerase II and promotes histone methylation of coding sequences, but the mechanism of Paf1C recruitment was unknown. We have shown that GCN4 recruits Paf1C in a manner dependent on PIC assembly and phosphorylation of Serine 5 in the C-terminal domain (CTD) of the largest subunit of RNA Polymerase II (Pol II) by KIN28. Importantly, we found that elongation factor SPT4 is also required for Paf1C occupancy at GCN4-induced genes and for Paf1C association with Serine 5-phosphorylated Pol II in extracts. Thus, it appears that SPT4 (or its partner in DSIF, SPT5) provides a platform on elongating Pol II for recruiting Paf1C following promoter clearance. Deletion of SPT4 reduces trimethylation of Lys4 on histone H3, demonstrating a new role for DSIF in this Paf1C-dependent function in transcription elongation.